U.S. patent number 4,568,488 [Application Number 06/570,075] was granted by the patent office on 1986-02-04 for reverse immunoaffinity chromatography purification method.
Invention is credited to Sylvia Lee-Huang.
United States Patent |
4,568,488 |
Lee-Huang |
February 4, 1986 |
Reverse immunoaffinity chromatography purification method
Abstract
One aspect of the present invention relates to a method for the
purification of a protein component of a biological fluid, said
method comprising: raising antibodies to impurities commonly
present in crude preparations of said components; preparing an
immunoadsorbent complex by linking said antibodies to a solid
adsorbent suitable for use in column chromatography; processing a
preparation of said component containing impurities through a
chromatography column containing said immunoadsorbent, thereby
causing selective adsorption of said impurities and exclusion of
said component in the effluent; and recovering said purified
component from said effluent. Another aspect of this invention is
directed to human urinary erythropoietin purified by the above
method.
Inventors: |
Lee-Huang; Sylvia (New York,
NY) |
Family
ID: |
24278110 |
Appl.
No.: |
06/570,075 |
Filed: |
January 11, 1984 |
Current U.S.
Class: |
530/397; 435/226;
514/15.3; 514/20.9; 514/7.7; 514/9.7; 530/380; 530/395; 530/399;
530/413 |
Current CPC
Class: |
C07K
14/505 (20130101); C07K 1/22 (20130101) |
Current International
Class: |
C07K
1/00 (20060101); C07K 1/22 (20060101); C07K
14/435 (20060101); C07K 14/505 (20060101); C01G
007/00 (); A61K 037/24 (); A61K 035/22 () |
Field of
Search: |
;260/112R,112B
;424/99,100,85,88,177 ;435/226 ;514/21 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
3994870 |
November 1976 |
Neurath et al. |
4172827 |
October 1979 |
Giaever |
4252902 |
February 1981 |
Fujii et al. |
4254095 |
March 1981 |
Fisher et al. |
4289689 |
September 1981 |
Friesen et al. |
4289690 |
September 1981 |
Pestka et al. |
4303650 |
December 1981 |
Takezawa et al. |
4332717 |
June 1982 |
Kanaoka et al. |
4361509 |
November 1982 |
Zimmerman et al. |
4377513 |
March 1983 |
Sugimoto et al. |
4397840 |
August 1983 |
Takezawa et al. |
4465624 |
August 1984 |
Chiba et al. |
|
Other References
J of Biol. Chem. 252, 5558-5564 (1977), Miyake et al. .
Experientia, vol. 29, p. 758, (1973), Sieber et al. .
Proc. Acad. Sci. USA, 74, 4633-4635 (1977), Spivak et al. .
Blood, 56, No. 4, 620-624 (1980), Lee-Huang. .
Blood, 52, No. 6, 1178-1188, Spivak et al. .
J. Lab. Clin. Med.-vol. 93, 40-53 (1979), Tuddenham et al. .
J. Lab. Clin. Med. vol. 101, 736-746 (1983, May), Rotblats et al.
.
Proc. Natl. Acad. Sci. USA, vol. 79, 1648-1652 (1982), Fulcher et
al. .
Scientific American, 66-74 (1980), Milstein, vol. 243..
|
Primary Examiner: Schain; Howard E.
Attorney, Agent or Firm: Darby & Darby
Government Interests
The United States Government has rights to this invention by virtue
of grant No. R01-HL21683 by the National Institute of Health,
Bethesda, Maryland.
Claims
What is claimed is:
1. A method for the purification of a protein component of a
biological fluid, said method comprising:
(a) raising antibodies to all of the antigenic substances other
than said protein, commonly present, as impurities, in a crude
preparation of said protein, and purifying said antibodies;
(b) preparing an immunoadsorbent complex by linking said antobodies
against said impurities to a solid adsorbent suitable for use in
column chromatography;
(c) processing a preparation of said protein containing said
impurities through a chromatography column containing said
immuoadsorbent, thereby causing selective adsorption of said
impurities in one step and exclusion of said protein in the
effluent; and
(d) recovering said purified protein from said effluent.
2. The method of claim 1 wherein said protein is a weakly
immunogenic protein compared to said impurities.
3. The method of claim 1, wherein said protein is present in said
biological fluid in minor quantities.
4. The method of claim 1, wherein said protein is selected from the
group consisting of weakly immunogenic proteins, glycoproteins,
hormones and enzymes present in said biological fluid in minor
quantities.
5. The method of claim 1, wherein said biological fluid is selected
from the group consisting of human plasma, human urine and human
urine concentrate.
6. The method of claim 1, wherein, prior to any of said steps, said
biological fluid is subjected to centrifugation to remove insoluble
impurities and to chromatography through a hydrophobic gel column
to remove the bulk of impurities present in said biological
fluid.
7. The method of claim 1, wherein: said step (a) comprises
immunizing laboratory animals with crude preparations of said
protein, said immunization resulting in simultaneous formation of
antibodies to said protein and to all of said impurities, and
separating said antigenic impurity antibodies from the antibodies
to said protein.
8. The method of claim 7 wherein said step (b) further comprises
preparing a second immunoadsorbent complex by linking said
antibodies to said protein to a second solid adsorbent suitable for
use in column chromatography, and, prior to said step (c),
processing said biological fluid through a second chromatography
column containing said second immunoadsorbent, thereby causing
selective exclusion of the bulk of the impurities contained in said
fluid, and employing the fraction of said biological fluid adsorbed
in said second column as the preparation of said protein in said
step (c).
9. The method of claim 8, wherein said protein is
erythropoietin.
10. A method for the purification of a ptorein component of a
biological fluid said method comprising:
(a) concentration said biological fluid;
(b) centrifuging said concentrate to remove insoluble impurities
present in said fluid;
(c) processing said centrifuged concentrate through a crosslinked
neutral gel chromatographic column, said gel containing a
hydrophobic group, whereby non-hydrophobic contaminants present in
said centrifuged concentrate are exluded and hydrophobic
constituents of said centrifuged concentrate are subsequently
recovered by elution from said column;
(d) raising antibodies to all of the antigenic substances other
than said protein commonly present, as impurities, in crude
preparations of said protein by injecting in laboratory animals a
quantity of a curde preparation of said protein sufficient to
induce an immune response in said animals, obtaining specific
anitsera from said animals and separating said antigenic impurity
antibodies from said specific antisera;
(e) linking immunoglobulins of said specific antisera, after
separation of said antigenic impurity antibodies therefrom, to a
first solid adsorbent suitable for use in column
chromatography;
(f) processing said constituents of said concentrate recovered by
elution in step (c) through said first immunoadsorbent, whereby the
bulk of the impurites remaining in said step (c) concentrate are
excluded in the effluent and recovering, as a product, the
remaining consitutents of said step (c) concentrate by elution;
(g) linking said antigenic impurity antibodies to a second solid
adsorbent support suitable for use in column chromatography;
(h) processing said step (f) product through said second column,
wherein the remaining impurites of said protein adsorb to their
antibodies and the protein is exclused in the effluent; and
(i) recovering said purified protein from said second column
effluent.
11. The method of claim 10, wherein said biological fluid is
urine.
12. The method of claim 11 wherein said protein is erythropoietin.
Description
FIELD OF THE INVENTION
The present invention relates to a method for purifying a protein
component of a biological fluid. More specifically, the present
invention relates to a novel immunoaffinity chromatographic method
for purifying a protein that could not be adequately purified by
conventional immunoaffinity chromatographic techniques. The
invention is particularly suitable for purifying erythropoietin,
but is equally applicable to purification of a great variety of
weakly immunogenic proteins, glycoproteins, hormones and enzymes
which are difficult to resolve from their contaminants.
BACKGROUND OF THE INVENTION
Human erythropoietin (Ep) is an acidic glycoprotein hormone with an
apparent molecular weight of 34,000 daltons. It is the primary
regulator of erythrocyte (red blood cell) production. Its known
major functions are promotion of erythroid differentiation and
initiation of hemoglobin synthesis, but it may also be involved in
stimulation of limited proliferation of immature erythrocyte
precursors.
An understanding of the mode of Ep action is of considerable
biological importance. Not only would it serve as a useful model
for studying the differentiation and development of mammalian
cells, but it would also be of great value in the diagnosis and
treatment of anemias. Although much research has been directed to
this area, progress has been slow due in part to the lack of pure
Ep. This is caused both by scant availability of starting materials
and by difficulties in purification.
Unavailability of sufficient quantities of pure Ep has also
hindered the development of Ep-specific monoclonal antibodies using
hybridoma techniques and the use of recombinant DNA technology in
the molecular cloning of Ep genes and the production of hybrid
cells which would produce human Ep gene products.
Ep circulates in the plasma space and is excreted in the urine at
very low concentrations under normal conditions. However, under
anemic or anoxic stress, Ep levels in the urine may increase
considerably. Thus, urine from severely anemic patients (e.g.,
patients with aplastic anemia, leukemia, or various
hemoglobinopathies) have been the sole source of human Ep, to date.
Not all anemic patients, however, exhibit increased urinary Ep
levels. Accordingly, monitoring of patients is necessary to
determine whether their urine will be useful as a source of Ep.
Moreover, once a patient responds to a therapeutic treatment, his
or her urinary Ep levels change rapidly, making it necessary to
seek a new Ep source. In addition, Ep must be purified from the
urine before it can be further used.
Many attempts have been made in various laboratories to purify
human Ep. The major difficulties with these attempts have been the
limited supply of starting material, and the incomplete resolution
of Ep from urinary contaminants. Early attempts to fractionate with
organic solvents and salts resulted in a distribution of activity
in several fractions. The fractions of higher activity have often
been obtained in low yield. Conventional chromatographic techniques
have been similarly limited in efficiency. Several purification
procedures have been reported. One such procedure described by
Espada, I., et al, Purification de Erythropoietina Urinaria Humana,
Acta Physiol., Lat. Am. 10:122-129, 1970, involved a ten-step
operation; briefly: (1) benzoic acid adsorption, (2) protein
precipitation, (3) ethanol precipitation, (4) heat treatment, (5)
Diethylaminoethyl(DEAE)-cellulose chromatography, (6)
hydroxylapatite adsorption, (7) 2nd DEAE-cellulose chromatography,
(8), (9), and (10) 1st, 2nd, and 3rd Sephadex G100 gel filtration.
This procedure gave a 323-fold purification with 18.5% yield. The
specific activity increased from 25 units/mg of protein in the
starting material to 8086 units/mg of protein in the final product
(units as defined below). According to these workers, this
procedure is efficient only when applied to large amounts of raw
material and when the starting material has an Ep titer of 20
units/mg or higher. The starting material used in the
above-described work was urine collected in Argentina from patients
afflicted with anemia due to hookworm infection.
Another procedure reported by Miyake, T, et al, Purification of
Human Erythropoietin, J. Biol. Chem. 252:5558-5564, 1977, consisted
of initial desalting on Sephadex G25, followed by seven steps,
namely: (1) DEAE batch elution, (2) p-aminosalicylate treatment and
phenol extraction, (3) ethanol fractionation, (4) DEAE agarose
column chromatography, (5) sulfopropyl-Sephadex chromatography, (6)
Sephadex G100 gel filtration, and (7) hydroxylapatite adsorption.
Again, this procedure requires large amounts of starting sample
with high initial specific activity. Seven million units with an
exceptionally high starting Ep titer of 91 units/mg of protein were
processed all at once. The final product had a specific activity of
70,400 units/mg of protein. This represented a purification factor
of 930 with 21% yield.
High Ep titer urine from aplastic anemic patients of unknown origin
collected in Kumanoto City, Japan was used as the starting
material. In the United States, however, it is impossible to have a
large supply of urine of such high Ep titer due to the practice of
giving anemic patients blood transfusions. Thus, it is impossible
to repeat this procedure on comparable starting material with
equivalent Ep titer. Quite different results and much lower
specific activity have been obtained when repeating this process on
a small scale with low Ep titer urine samples collected in the
U.S.A.
Furthermore, each of the above procedures requires constant use of
large amounts of benzoic acid and phenol. The former is toxic, and
the latter a known mutagen; they are thus deleterious to Ep
research goals.
Aside from the problems due to the extremely low initial content of
Ep in urine, purification of the hormone is quite difficult to
achieve because it is contaminated with many urinary impurities
with similar physiochemical properties. Many of the existing
purification procedures are based on either conventional charge and
size separations, or sugar-specific affinity to lectin derivatives.
A simple prior group separation on the basis of a different and
independent property, hydrophobicity, proved important for the
elimination of contaminating impurities from Ep with similar size
and charge as well as similar monosaccharide content. Use of
hydrophobic interaction chromatography (HIC) in Ep purification,
has been reported by Lee-Huang, S. A New Preparative Method for
Isolation of Human Erythropoietin with Hydrophobic Interaction
Chromatography, Blood 56: 620-624, 1980.
Immunoaffinity chromatography is highly specific and effective for
the purification of many macromolecules. However, in the absence of
sufficient quantities of pure Ep as the immunogen for the
production and/or purification of Ep-specific antibodies, the
potential of conventional immunoaffinity becomes limited. Even if
highly purified Ep is used for the immunization of antibody
producing animals, these animals frequently generate large amounts
of antibodies against minor contaminants, especially when the main
antigen is a weak immunogen, as is the case with Ep. Conventional
immunoaffinity chromatography can thus only yield a preparation as
pure as the original antigen, since antibodies to the contaminants
also immunoabsorb their antigens.
The present invention involves a novel and simple immunoaffinity
technique for use in Ep purification. The experimental results
showed excellent potential and general applicability of the
procedure. This novel procedure is especially well suited for
initial processing of crude starting material of moderate Ep titer.
In bypassing many steps, unnecessary handling of the sample is
eliminated, and the yield is increased accordingly. The present
specification includes a description of a systematic investigation
of some of the important parameters for high resolution and good
recovery. By the combination of HIC, Direct Immunoaffinity
chromatography (DIAC), and Reverse Immunoaffinity Chromatography
(RIAC) in particular, a purification factor of 35,000 fold with 59%
yield has been achieved. The specific activity increased from 0.91
units/mg of protein in the starting material to 32,000 units/mg of
protein in the final product. This procedure is simple, rapid, and
effective, and is suitable for the processing of low and high Ep
titered urine in large or small quantities. Some of the starting
material was supplied by the National Heart, Lung, and Blood
Institute. Additional urine samples were collected from various
hospitals in New York City from patients suffering from disorders
including aplastic anemia, hemolytic anemia, leukemia and various
hemoglobinopathies.
OBJECTS OF THE INVENTION
Accordingly, it is an object of this invention to provide a method
for the purification of proteins, and particularly weakly
immunogenic proteins present in biological fluids in minor
quantities.
It is another object of this invention to provide a convenient
method for the purification of such proteins in high purity and
high yield at relatively low cost.
Another object is to provide a method for simple and rapid
purification of erythropoietin.
It is a further object of this invention to provide a method for
purification of biological fluid protein components which bypasses
the need of an initial supply of pure protein and pure antibody of
such protein.
It is yet another object of this invention to provide a method for
purification of biological fluid protein components with an
efficiency not heretofore attainable with prior art methods, while
preserving the activity of such proteins.
It is still another object of this invention to prepare highly
purified erythropoietin in sufficient quantities for the
development of its diagnostic and therapeutic applications.
Another object of this invention, is to prepare purified active
erythropoietin from a biological fluid, suitable for use in
development of Ep-specific monoclonal antibodies, and in quantities
sufficient for such use.
These and other objects of this invention will be apparent to those
skilled in the art in light of the present description, appended
drawings and accompanying claims.
SUMMARY OF THE INVENTION
One aspect of the present invention relates to a method for the
purification of a protein component of a biological fluid, said
method comprising:
raising antibodies to impurities commonly present in crude
preparations of said component;
preparing an immunoadsorbent complex by linking said antibodies to
a solid adsorbent suitable for use in column chromatography;
processing a preparation of said component containing impurities
through a chromatography column containing said immunoadsorbent,
thereby causing selective adsorption of said impurities and
exclusion of said component in the effluent; and
recovering said purified component from said effluent.
Another aspect of this invention is directed to active human
urinary erythropoietin purified by the above method.
BRIEF DESCRIPTION OF THE DRAWING
The present invention is illustrated in the accompanying drawings
in which:
FIG. 1 is a drawing of photographs of: (a) a Sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) pattern on slab gel,
(b) an isoelectric focusing pattern on a disc gel, and (c) an
electrophoretic pattern in a non-dissociating disc gel, said
patterns being of human urinary Ep purified according to the method
of the present invention.
FIG. 2 is a drawing of photographs of electrophoretic patterns of
SDS-PAGE slab gel (a) by silver staining and (b) by autoradiography
of .sup.125 I- labeled Ep, said patterns being of human urinary Ep
purified according to the present invention.
FIG. 3 is a plot of the spectrophotometric absorbance and
biological activity pattern of Ep prepared by (a) direct
immunoaffinity chromatography (FIG. 3A) and (b) subsequent reverse
immunoaffinity chromatography of the (a) product in accordance with
the present invention (FIG. 3B).
FIG. 4 is a flow chart outlining the various steps of the present
invention .
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described further below with particular
reference to purification of erythropoietin from concentrated urine
in accordance with preferred embodiments. Such specific description
does not detract, however, from the general applicability of the
present method to purification of other protein constituents of
biological fluids. The method of the present invention may be used
in the purification of other weakly immunogenic proteins,
glycoproteins, enzymes and hormones, whose contaminants are
difficult to remove by conventional methods because, during
immunization for the purpose of obtaining antibodies to such
proteins, corresponding antibodies to the main antigen and its
contaminating impurities are concurrently produced. Of course, as
those skilled in the art will readily appreciate, the procedures
for the resolution of these antibodies may differ according to the
substance to be purified. For example, if the protein desired to be
purified is a glycoprotein, purification of antibodies may be
carried out on a lectin-glycoprotein column; if it is an enzyme,
purification may be conducted on an enzyme substrate
analogue-enzyme column; if it is a metal-containing protein,
purification may be accomplished on a
Thiopropyl-Sepharose-metal-containing protein column, or an
iminoacetic acid Sepharose-metal-containing protein column. If it
is a sulfhydryl containing protein, an organomercurial Sepharose
column may be used, since the organomercurial readily forms a
covalent mercaptide with free sulfhydryls. The solid supports
mentioned here are not restricted to Sepharose. Agarose or other
forms of gel matrix are also appropriate. In addition, the eluant,
buffer, and other media used, should be selected in accordance with
the affinity characteristics of the substance to be purified and
with due consideration given to the differential affinity between
its immunospecific and biospecific ligands where applicable, as is
well known in the art.
According to the present method, unconcentrated or concentrated
urine from severely anemic patients can be used as the raw
material. Starting samples, are preferably first centrifuged to
eliminate insoluble material and purified preferably by hydrophobic
interaction chromatography (HIC), as described by Lee-Huang, S.: A
New Preparative Method for the Isolation of Human Erythropoietin
With Hydrophobic Interaction Chromatography, Blood 56:620-624,
1980, in order to remove the bulk of urinary contaminants and
permit more efficient and repeated use of the immunoadsorbents. HIC
involves processing of the raw material through a crosslinked
neutral gel chromatographic column wherein the gel contains a
hydrophobic group. Phenyl-Sepharose CL4B is particularly preferred
because it provides a strong yet easily reversible binding with Ep.
Octyl-Sepharose may also be used, but Ep elution therefrom is less
complete. The specific activity of Ep obtained from this step
depends on the potency of the starting material but generally
ranges between about 115 and 250 units per mg of protein. The yield
is usually about 80%. One unit of Ep is defined as the activity
contained in 0.5 mg of the second International Reference
Preparation of Human Urinary Erythropoietin (IRP) (obtained from
the World Health Organization, International Laboratories of
Biological Standards, Hampstead, London, England), or one-tenth of
the contents of one ampule of this preparation.
The HIC-purified material can be used as the immunogen to raise
antibodies to Ep (hereinafter designated as "Anti-Ep") and its
common contaminating impurities (hereinafter designated as
"Anti-I"). This can be conveniently performed in a single
immunization using antibody-producing laboratory animals. The
immunization is carried out in accordance with methods well known
in the art and, in the case of Ep or other weak immunogens, it
preferably includes several booster injections in addition to the
initial injection. Anti-Ep titers are determined by the in vivo
exhypoxic polycythemic mouse bioassay described by Camiscoli, J. F.
and Gordon, A. S.: Bioassay and Standardization of Erythropoietin
in Gordon, A. S. (Ed.) Regulation of Hematopoiesis, Meredith Corp.,
New York, 1970 pp 370-396.
Polycythemia is induced in mice by hypobaric hypoxia. In order to
keep a high protein concentration and thus stabilize the Ep
activity, Ep samples for assay are made up in a buffered albumin
solution. Samples are injected into mice posthypoxia,
intraperitoneally. Ep activity is measured by its stimulation of
.sup.59 Fe incorporation in red blood cells. .sup.59 Fe
incorporation is determined in a gamma counter. The results are
compared to those obtained using the second IRP from WHO. Anti-Ep
titers are determined by assaying for ability to neutralize
Ep-stimulated .sup.59 Fe incorporation in red blood cells.
The immunized laboratory animals are then finally bled. Antisera
from the bleedings after the last injection are isolated, assayed
for anti-Ep titers, and purified by immunoaffinity chromatography
to eliminate non-immunoglobulins. The rabbit antisera are processed
through a Sepharose 4B column to which goat-(anti-rabbit) Igs have
been covalently linked. The non-immunoglobulins are excluded from
the column, while the specific Igs are eluted with, e.g., 3M sodium
thiocyanate (NaSCN) or 0.2M acetic acid.
The thus obtained specific immunoglobulin preparation is treated to
separate Anti-Ep from Anti-I. For this purpose, a highly purified
Ep preparation is preferably used. However, the present invention
does not require pure Ep for antibody preparation and/or
separation. Partially purified Ep (or other partially purified
antigen), prepared according to conventional methods, is adequate
for carrying out the method of the present invention.
The antibody separation may be preferably accomplished by a new
principle and procedure which employs reversible binding of antigen
to a supporting matrix and thus permits subsequent recovery of
valuable Ep (or other antigen) after it is used in the Anti-Ep (or
other antibody) purification, without substantial loss of
activity.
The antibody separation procedure utilizes the fact that Wheat germ
Lectin-Sepharose 4B (WGLS) columns coated with purified Ep have
differential affinity for their biospecific and immunospecific
ligands. The procedure involves four steps:
1. Purified Ep is bound to WGLS to produce a WGLS-Ep complex. Ep
binds tightly to WGLS due to interaction of its
N-acetyl-glucosaminyl residues with the wheat germ lectin.
2. The affinity purified rabbit immunoglobulins are processed
through a WGLS-Ep column: Anti-Ep binds to the WGLS-Ep complex,
while Anti-I does not, but is excluded in the effluent and set
aside for further use. Of course, since the original purified Ep,
that was used to coat the WGLS column in Step 1, was not
homogeneous, its impurities will also be carried over to the WGLS
column of Step 1 and, consequently a small fraction of the Anti-I
will bind to the WGLS-Ep column of Step 1. This was the shortcoming
of conventional immmunoaffinity techniques which the present
invention has overcome, as will be described below. Anti-Ep bound
to the WGLS-Ep complex is eluted and preferably processed again
through a regenerated WGLS-Ep column to insure complete resolution
of Anti-Ep/Anti-I immunoglobulins. The Anti-I-containing eluents
from the first and the second separation are pooled and Anti-I are
recovered therefrom.
3. Since the affinity between the constituents of the immune
complex (Ep-(Anti-Ep)) is lower than the affinity between Ep and
the sugar-lectin complex (WGLS-Ep), Anti-Ep from WGLS-Ep-(Anti-Ep)
can be selectively eluted using a weak acid or a dissociation
reagent. The ability of WGLS to bind Ep both at low pH and under
dissociating conditions makes WGLS a useful adsorbent for Anti-Ep
purification and at the same time enables recovery of the valuable
Ep (see step 4 below). The thus recovered Anti-Ep is separated from
the eluent (e.g., by dialysis) lyophilized, and stored frozen for
subsequent use.
4. Ep can be recovered from WGLS-Ep, once Anti-Ep has been eluted,
by further elution, preferably with N-acetylglucosamine or
N,N-diacetylchitobiose. This is not possible under conventional
immunoaffinity procedures since, normally, the immunoadsorbent is
irreversibly coupled to the supporting matrix and cannot be
recovered. When the supply of the antigen (used as the
immunoadsorbent) is limited, the recovery of such materials is a
very valuable saving. Alternatively, the column of WGLS-Ep can be
regenerated and can be reused.
The thus recovered Anti-Ep and Anti-I are separately covalently
linked to CNBr-activated Sepharose 4B. The coupling procedure has
been generally described by Axen, R. et al "Chemical Coupling of
Peptides and Proteins to Polysaccharides by Means of Cyanogen
Halides" Nature, 214:1302-1304, 1967. The Sepharose-(Anti-Ep) and
Sepharose-(Anti I) so prepared are used in column form for the
direct immunoaffinity chromatography (DIAC) and reversed
immunoaffinity chromatography (RIAC) purification of Ep.
The Ep purified by HIC is further purified by DIAC on a
Sepharose-(Anti-Ep) column. This purification results in exclusion
of the majority of contaminants from the column, which are carried
off in the effluent, while Ep is retained on the column. It is
important to note, however, that at this stage some antibodies to
some minor impurities will be present in the Sepharose-(Anti-Ep)
column because of the lack of homogeneous Ep in the immunoaffinity
purification of the Anti-Ep. This is the intrinsic limitation of
any conventional direct immunoaffinity technique.
Ep from the Sepharose-(Anti-Ep) column is eluted with an
appropriate buffer. Choice of buffer is important in preserving Ep
activity. For example, commonly used immune complex dissociating
acidic buffers or chaotropic ions (such as glycine hydrochloride
buffer or sodium thiocyanate) inactivate Ep, while simple alkali
gives incomplete desorption. The present inventor has found that
inclusion of 10-20% of a polarity reducing agent (such as glycerol
or another common 1,2-glycol) and a dissociation agent (such as
guanidine hydrochloride or urea) in an alkaline eluant (such as
NaOH) facilitates effective release of Ep from the immunoadsorbent
while preserving Ep activity. Preferred are ethylene glycol and
guanidine hydrochloride, which can be easily removed and which
appear to have no detrimental effect on Ep activity.
The thus eluted Ep is dialyzed (preferably immediately and
thoroughly) against water and sodium phosphate buffer. Under these
conditions, DIAC is very efficient, offering a high purification
factor (usually about 169-fold over HIC) and a high yield (usually
about 80% or higher). However, the main limitation of DIAC is the
impurities in the original Ep preparation. The antibodies against
these impurities are carried over in the purification system and
immunoadsorb their antigens in the Sepharose-(Anti-Ep) column. As a
consequence, the purity of the DIAC product cannot exceed that of
the original Ep used in preparation of the WGLS column (Step 1) for
antibody purification.
At this point further purification is accomplished with another
Sepharose column coupled with Anti-I. While Anti-Ep contains only a
minor fraction of antibodies to the impurities, Anti-I consists of
the bulk of these antibodies. Thus, the Sepharose-(Anti-I) column
will be able to provide sufficient antibody sites to bind
substantially all the impurities contained in the DIAC-purified
Ep.
Upon loading DIAC-purified Ep onto a Sepharose-(Anti-I) column, the
trace impurities are retained in the column due to the formation of
specific immune complexes with their corresponding antibodies,
which are present in great excess on the column, whereas pure Ep is
selectively excluded in the effluent. This step affords preparation
of Ep which is purer than the original antigen. Such efficiency is
not attainable with other conventional immunoaffinity techniques.
This immunoaffinity chromatography step wherein the impurities are
bound to their antibodies, while the valuable protein is excluded
in the effluent, is referred to as Reverse Immunoaffinity
Chromatography (RIAC).
The impurities removed in the reverse immunoaffinity step are a
constant set of residual urinary contaminants; and they have been
copurified with Ep in many separation techniques, and are therefore
fairly uniform from batch to batch. Thus, crude urine from a source
different from that employed to generate the antisera can be
effectively purified by the HIC-DIAC-RIAC procedure. The amount of
Anti-I required for immunoadsorption of these minor impurities of
DIAC-purified Ep is small relative to the total capacity of the
Sepharose-(Anti I) column. Furthermore, since reverse
immunoaffinity chromatography immunoadsorbs only the contaminating
impurities, no desorption of Ep is required, thus minimizing
manipulation of valuable samples and increasing yield accordingly.
The impurities retained on the column can be subsequently
dissociated from the immunoadsorbent by eluting with an appropriate
acidic eluent. The column is thus regenerated and ready for
subsequent use.
The DIAC-RIAC purified Ep can be tested for homogeneity by
attempting further purification using conventional purification
techniques (preferably chromatographic techniques and/or gel
filtration), and assayed for biological activity.
The DIAC-RIAC purified Ep is further tested for homogeneity and
characterized by electrophoretic techniques, such as gel
electrophoresis, isoelectric focusing, and disc electrophoresis in
non-dissociating systems according to well-known methods described
by: (a) Laemmli, U. K.: Cleavage of Structural Proteins During the
Assembly of the Head of Bacteriophage T.sub.4, Nature 227: 680-685,
1970; (b) Catsimpoolas, N. et al (Ed.); Biological and Biomedical
Application of Isoelectric Focusing, New York, Plenum Press, 1977,
and (c) Davis, B. J.: Disc Electrophoresis-II: Method and
Application to Human Serum Protein, Ann. N.Y. Acad. Sci.
121:404-427, 1964.
The following examples serve further to illustrate the present
invention, but not to limit its scope.
Materials: Phenyl-Sepharose CL4B, ConA-Sepharose 4B, Wheat germ
Lectin-Sepharose 6MB, CNBr Activated Sepharose 4B Sephadex G100
were obtained from Pharmacia Laboratories, Inc., (Piscataway, N.
J.) Guanidine hydrochloride (ultra-pure) was obtained from
Schwartz-Mann Biochemicals (Spring Valley, N. Y.) Ethylene glycol
and N-acetylglucosamine were from Sigma Chemical Company (St.
Louis, MO.). All other chemicals were from Fisher Scientific
Company (Fairlawn, N. J.), except when otherwise specifically
indicated.
Concentration of all column eluates was carried out at 4.degree. C.
using an Amicon ultrafiltration apparatus with YM10 membrane unless
otherwise specified.
EXAMPLE 1
Initial purification of Ep
Urine from selected patients with elevated Ep titers (as determined
by the exhypoxic polycythemic mouse bioassay, see below) was
obtained from the National Heart, Blood and Lung Institute as well
as from physicians in several hospitals in New York City. The
patients suffered from disorders including aplastic and/or
hemolytic anemia, leukemia, and various forms of
hemoglobinopathies.
The starting urine sample was first concentrated by an Amicon DC-2
concentrator/dialyzer (Amicon Corp., Lexington, Mass.) using a H1
DP10 hollow fiber cartridge (10,000 molecular weight exclusion) in
order to remove low molecular weight contaminants while
simultaneously concentrating. The flow rate of the effluent was
initially adjusted to 50 ml/min. The concentrates were dialyzed
against distilled water in the same apparatus. The dialyzed samples
were then lyophilized and stored in sterile containers at
-70.degree. C. Between 130 and 160% of Ep activity was routinely
recovered by this process, suggesting that Ep inhibitors with
molecular weights less than 10,000 are present in the crude urine,
and that they are removed by the H1DP10 hollow fiber.
The concentrates were then processed by HIC on a Phenyl-Sepharose
CL4B (PS) column. Generally about 3,000 to 6,150 units of pooled
lyophilized urine concentrate were dissolved in 34 ml of the
starting buffer (10 mM sodium phosphate/ 4 M NaCl, pH 6.8) by
gentle stirring. Any insoluble material was removed by
centrifugation at 10,000.times.g for 30 min. The clear supernatant
solution was loaded onto a PS column (2.5.times.81.5 cm, bed volume
400 ml) previously fully equilibrated with the same starting buffer
(by matching of the buffer refractive index to that of the column
effluent). 25 ml fractions were collected at a flow rate of 1.5
ml/min. Unbound impurities were washed with the buffer until
effluent absorbance at 280 nm (A.sub.280 nm) was zero. The column
was then eluted with 10 mM sodium phosphate saline buffer (0.5 M
NaCl; pH 7.1) to further eliminate urinary contaminants. Ep
activity was eluted with 10 mM NaOH containing 20% ethylene glycol
and 4M guanidine hydrochloride. The fractions showing Ep activity
were pooled. The resulting solution was concentrated by
ultrafiltration using an Amicon YM10 membrane. The concentrated
sample was dialyzed against PBS (5 mM sodium phosphate containing
0.15 M NaCl) to remove residual guanidine hydrochloride.
Approximately 204 mg of product was obtained from the pooled
fractions of every ten PS columns, with a mean specific activity of
124 units per mg. representing 136-fold of purification with 82%
yield. This material was stored at -70.degree. C., until sufficient
quantities were accumulated for the following usages: it was used
as a source of partially purified Ep (Example 3) for antibody
purification (Example 4) and as a source for purification according
to the present method (Example 6 et. seq.). A particular sample
containing 240 units of Ep per mg of protein was used for
immunization, described in Example 2 below.
EXAMPLE 2
Immunization of Antibody Producing Animals
Female New Zealand white rabbits (2-2.5 kg initial body weight)
were used for immunization. The sample selected from Example 1 (5
mg/ml, 240 u/mg) was emulsified with an equal volume of Freund's
adjuvant (obtained from Difco Laboratories, Detroit, Mich.) either
complete for primary injection or incomplete for booster
injections. One ml of this mixture was injected subcutaneously at
multiple sites each time. Booster injections (a total of 3) were
given every six weeks and the rabbits were bled two weeks after
each boost. A total dose of 2400 units of Ep were given to each
animal from the primary and booster injections. Anti-Ep titers were
determined by the in vivo exhypoxic polycythemic mouse bioassay, as
follows:
CF- 1 virgin female mice (22-25 g body weight) from Charles River
Laboratories (Boston, Mass.) were used. The animals were exposed to
0.4 atm for 219 hrs (19 hr/day) in a decompression chamber to
induce hypobaric hypoxia. Ep and Anti-Ep samples (0.5 ml/injection)
were made up in a solution of 0.5% albumin in 0.15 M NaCl and
injected intraperitoneally 72 hrs posthypoxia. Antisera were added
to Ep samples in various amounts and incubated at 37.degree. C. for
2 hours and then 4.degree. C. overnight before injection. An amount
of 0.5.mu. Ci .sup.59 FeCl.sub.3 in 0.1 ml of saline/albumin was
administered to the mice intravenously on the fifth day. Ep
activity was measured by its stimulation of .sup.59 Fe
incorporation into the circulating red cells (obtained by cardiac
puncture) 48 hr after .sup.59 Fe injection. Differences in Ep
activity observed in mice injected with Ep-antibody mixtures
provided a measure of Anti-Ep activity. Percent incorporation of
.sup.59 Fe was determined on 0.5 ml of blood sample in a Beckman
Gamma 4000 counter (Beckman, Inc., Palo Alto, Calif.). The second
International Reference Preparation of Human Urinary Ep (WHO) was
used as a standard. Ep potency was expressed in units per mg of
protein. Hematocrit factors were determined in duplicates by the
microhematocrit method, using an Autocrit II centrifuge from Clay
Adams Division, Becton Dickinson and Company (Parsippany, N. J.)
Results from animals with a hematocrit factor of less than 0.52
were discarded. Dose level means were based on quadruplicates.
Anti-Ep content of the antisera ranged from 10-20 units per ml
after the first boost and rose to 100-200 units per ml after the
last boost.
Blood samples were obtained from the ear vein and allowed to clot
at 4.degree. C. for 30 minutes. The antiserum was collected by
centrifugation at 10,000 g for 30 min, and purified by an affinity
column (0.9.times.15 cm, bed volume 9 ml) of goat Anti-rabbit
immunoglobulins coupled to Sepharose 4B to eliminate the
non-immunoglobulin proteins.
Goat anti-rabbit immunoglobulins (Igs) were obtained from Miles
Laboratories (Elkhart, Ind.) and coupled to CNBr activated
Sepharose 4B as described in Example 5. The coupled material
contained about 25 mg Igs/ml of Sepharose. Ten ml of the rabbit
antisera were applied to a 9 ml column of goat Anti-rabbit Igs -
Sepharose 4B. The column was washed with PBS and the
nonimmunoglobulin proteins were excluded from the column in this
fraction. The antibodies bound on the columns were eluted with 0.2M
acetic acid. Routinely, 30 to 35 mgs of Igs were obtained per ml of
antiserum. The affinity purified rabbit Igs contain both Anti-I and
Anti-Ep. They were dialyzed against distilled water, lyophilized
and stored frozen at -70.degree. C.
EXAMPLE 3
Purification of Ep for Use in Antibody Purification
The Ep preparation of Example 1 was further purified successively
by: lectin affinity chromatography on a Con A-Sepharose column and,
subsequently on a Wheat germ Lectin-Sepharose (WGLS) column,
followed by adsorption chromatographgy on hydroxylapatite and,
finally, gel filtration on Sephadex G100.
(1) Lectin Affinity Chromatography on Con A-Sepharose. The Ep
preparation from Example 1 was dialyzed against buffer I (PBS
containing 0.1 mM of each MgCl.sub.2, MnCl.sub.2, and CaCl.sub.2,
pH 7.1) and applied to a column of Con A-Sepharose 4B which was
previously equilibrated with the same buffer. The column was washed
with buffer I. Ep activity was recovered in the unretained
material. The preferred ratio of mg protein loaded to bed volume
(ml) is 1:2.
(2) Lectin Affinity Chromatography on Wheat Germ Lectin-Sepharose.
The excluded material from the Con A-Sepharose 4B column was loaded
directly onto a WGLS 6MB column (at a 1:1 ratio of mg protein to ml
bed volume) which was previously equilibrated with PBS. The column
was washed with PBS until the A.sub.280 nm of the effluent reached
zero. Ep activity was eluted with buffer II (PBS containing 0.1 M
N-acetylglucosamine (NAGA)). The active fractions were pooled and
concentrated to 1 ml. The buffer of the sample was then changed to
0.5 mM sodium phosphate buffer, pH 7.1 (buffer III).
(3) Adsorption Chromatography on Hydroxylapatite. The Ep sample
from the previous step was loaded onto a hydroxylapatite column,
(at a 4:5 ratio of mg protein to ml bed vol) previously
equilibrated with buffer III. The column was washed with the same
buffer until the A.sub.280 nm of the effluent reached zero. It was
subsequently eluted stepwise with varying molarities of sodium
phosphate buffers, pH 6.8, and 1 ml fractions were collected. Ep
activity was found in the 2 mM eluate. The fractions containing Ep
activity were pooled and concentrated to 0.3 ml.
(4) Gel filtration on Sephadex G100. The Ep containing fraction
from the previous step was applied to a Sephadex G100 column
(0.5.times.100 cm, bed volume 19.6 ml) in buffer IV (2 mM sodium
phosphate, pH 7.1). Gel filtration was carried out in the same
buffer at a flow rate of 3 ml/hr. Fractions of 0.6 ml were
collected and Ep activity was eluted between 0.46 to 0.56 column
bed volume. The active fractions were pooled, concentrated,
lyophilized and stored frozen at -70.degree. C. for use in Anti-Ep
purification as discussed in Example 4.
In summary, the steps described in this Example 3 enabled the
production of highly purified Ep. A mean specific activity of
20,535 units/mg of protein was obtained for the final product,
corresponding to an overall Ep purification of 22,566 fold. The
overall yield ranged from 21% to 36%.
It is important to note that the combination of DIAC and RIAC as
described in this patent application would achieve a 35,299-fold
overall purification with an overall yield of 59%. This enrichment
in Ep yield and improvement in purification cannot be accomplished
by previously existing conventional techniques.
The purification method outlined in this Example 3, although
preferred, is not essential. Relatively pure Ep, however, would
have also been suitable.
EXAMPLE 4
Fractionation of the Antibodies of Example 2
A 2.5 ml column (0.8 .times.5 cm) of WGLS was equilibrated with PBS
and 1.5 mg of the Ep obtained in Example 3 was loaded onto the
column. The flowthrough was recycled six times to ensure complete
binding of Ep to WGLS. 140 mg of the purified antisera obtained at
the end of Example 2 were dissolved in 4 ml of PBS and applied to
the WGLS-Ep column. The column was washed with PBS until A.sub.280
nm reached zero. Anti-I was excluded from the column while Ep-bound
Anti-Ep was retained on the column. The effluent, containing
Anti-I, was set aside, and Anti-Ep was eluted from the column with
0.2M acetic acid.
The Anti-Ep and Anti-I were recycled separately on a regenerated
WGLS-Ep column to ensure maximum resolution. The pooled Anti-Ep
fractions (5 mg) and Anti-I fractions (134 mg) were dialyzed
separately against 4 mM NaHCO.sub.3, pH 8.2, lyophilized and stored
at -70.degree. C. until sufficient quantities were accumulated for
covalent coupling to Sepharose 4B.
The WGLS-Ep column was washed with 10 column volumes of PBS, and
the bound Ep was recovered by eluting the column with 0.1M
N-acetylglucosamine (NAGA). The specific activity of the recovered
Ep was not detectably different from that of the original Ep loaded
onto the WGLS column, indicating that no inactivation had occurred.
Alternatively, after washing with PBS, the column may be reused as
needed.
EXAMPLE 5
Coupling of antibodies to adsorbent
CNBr-activated Sepharose 4B was acid-swollen in 1 mM HCl for 30
min. at a ratio of 1 g of gel per 300 ml of acid, The swollen gel
was then washed on glass filter with 200 ml of 1 mM HCl, six times.
The Anti-Ep and Anti-I from Example 4 were then separately
dissolved in 0.1 M NaHCO.sub.3, pH 8.2, containing 0.5M NaCl to a
protein concentration of 25 mg/ml. 10 ml of each was then mixed
separately with an equal volume of the swollen gel in a 15 ml
polypropylene sterile tube. The mixture was rotated end-over-end on
an automatic nutator (American Hospital Supply Corp., Model R485-10
Evanston, ILL.) at 4.degree. C. overnight. To remove the unbound
material, the resulting mixture was filtered on glass filter
(porosity G3) and washed with the above coupling buffer. The
remaining active groups were blocked by reacting with 1M
ethanolamine at pH 8.2 for 16 hrs at 4.degree. C. with gentle
rotation.
Noncovalently adsorbed proteins were removed by four cycles of
washing: each cycle involved washing at pH 4.0 with 0.15M sodium
acetate buffer followed by washing at pH 8.2 with 0.1M sodium
borate buffer (both buffers containing 0.5M NaCl). Typical coupling
by this method yielded 94-96% of coupled protein in both Anti-Ep
and Anti-I samples.
EXAMPLE 6
Direct Immunoaffinity Chromatography
Material accumulated from Example 1, in 8 ml of buffer V (0.1M
sodium phosphate buffer, pH 7.5) at 25.5 mg/ml containing 25,378
units of Ep was subjected to direct immunoaffinity chromatography
(DIAC) on a Sepharose-(Anti-Ep) column (10 ml; 0.9.times.16 cm)
prepared using the appropriate complex of Example 5. The column was
washed with the same buffer until A.sub.280 nm of the effluent
reached zero. The majority of impurities were excluded from the
column and Ep was retained. 2 ml fractions were collected. The bulk
of the impurities was excluded in fractions 6-30 (FIG. 3A, peak 1,
wherein the continuous line designates absorbance at 280 nM and the
broken line designates Ep activity). Ep was eluted with buffer VI
(10 mM NaOH containing 20% ethylene glycol and 4M guanidine
hydrochloride, pH 10.4), as indicated by the arrow on the Figure.
Fractions 126-130 (peak 2 on FIG. 3A) contained Ep. The fractions
were dialyzed immediately and thoroughly against water and then
buffer V. Each fraction was assayed for activity and its A.sub.280
nm was measured. The active fractions were pooled and concentrated
to about 1 mg/ml for further purification.
EXAMPLE 7
Reverse Immunoaffinity Chromatography
The product of Example 6 (0.99 mg of Ep material/ml buffer V) was
applied to the Sepharose-(Anti-I) column (bed vol. 10 ml;
0.9.times.16 cm) and one ml fractions were collected, (shown in
FIG. 3B). The contaminating impurities were adsorbed on the column
by their specific antibodies. Pure Ep was obtained in the effluent
in fractions 11-18 (peak 1). Each fraction was assayed for Ep
activity and monitored for absorbance at 280 nm. The active
fractions were pooled and concentrated. This step offered effective
and specific removal of the trace residual contaminants. These
impurities copurify with Ep and are otherwise difficult to
eliminate by conventional separation methods. A final specific
activity of 32,122 u/mg was obtained with an overall purification
of 35,299 fold and 1.53-fold over the DIAC step. The overall yield
was 59% and the yield of the last step was 88% from DIAC.
The column was regenerated with 0.2M acetic acid (as indicated by
the arrow in FIG. 3B). The impurities were collected in fractions
70-76 (peak 2). The column was then equilibrated with buffer V, and
stored in said buffer containing 0.02% sodium azide at 4.degree.
C.
EXAMPLE 8
Test of Homogeneity of DIAC-RIAC Purified Erythropoietin
Protein concentration in samples was determined by a method
described in Bradford, M. M. "A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding." Anal. Biochem. 72:248-254,
1976.
Further processing of this Example 7-purified Ep material on one or
more of hydroxylapatite, Con A-Sepharose, Wheat germ
Lectin-Sepharose 6MB, or Sephadex G100 following the procedures
described in Example 3, did not increase the specific activity of
the Ep. Homogeneity of the DIAC-RIAC-purified Ep was tested by
polyacrylamide gel electrophoresis (SDS-PAGE) as follows:
Polyacrylamide gels were prepared according to Laemmli, supra,
containing 10% by weight acrylamide, and 0.24% by weight
N,N'-bis-methylene acrylamide in 0.375 M Tris-HCl, pH 8.8, sodium
dodecyl sulfate (0.1%), tetramethylethylenediamine (0.033%) and
ammonium sulfate (0.05%). The Ep preparation was treated with an
equal volume of 2.times. sample buffer containing 0.125 M Tris-HCl,
pH 6.8, 4% SDS buffer, 20% glycerol, and 10% 2-mercaptoethanol.
Electrophoresis was carried out at a constant voltage of 50 to 100
V for 8 to 4 hours. The gels were fixed and stained with 0.125%
coomassie brilliant blue dye in 50% methanol and 10% acetic acid.
The results are shown in FIG. 1A. For the sample from Example 7 (40
.mu.g) lane 3, a single band was obtained with a molecular weight
of 34,000 daltons. Lanes 1 and 2 are Ep preparations from Examples
3 and 6 (40 .mu.g each).
Isoelectric focusing was carried out in 7.5% acrylamide and 0.25%
bisacrylamide gel in the presence of 1% carrier ampholytes, pH
3-10. Sulfuric acid (0.2%) and ethylenediamine (0.4%) were used as
anodic and cathodic electrolytes, respectively. Electrofocusing was
carried out at room temperature. A maximum current of 2 mA/gel was
maintained by gradual increase of the voltage up to 200 V. The gels
were stained in 0.2% bromophenol blue in ethanol - H.sub.2 O -
acetic acid (50:45:5) and destained in ethanol - H.sub.2 O - acetic
acid (30:65:5). A single component with an isoelectric point at 4.1
was obtained (FIG. 1B, 15 .mu.g).
Finally, gel electrophoresis in a non-dissociating system was
carried out in a 7.5% polyacrylamide (0.24% bisacrylamide) gel in
Tris-glycine buffer 0.0426M Tris base (0.0242M glycine, pH 9.6) at
a constant current of 2.5 mA per gel column, according to Davis, B.
J.: Disc Electrophoresis - II: Method and Application to Human
Serum Proteins, Am. N.Y. Acad. Sci. 121:404-427, 1964. A single
band as seen on FIG. 1C was observed (20 .mu.g sample load).
The purity of Ep prepared in Example 7 was further examined by two
other methods: (a) silver stain of the SDS-PAGE sample of Ep, and
(b) radioiodination of Ep and autoradiography of the SDS-PAGE
sample of .sup.125 I-labeled Ep. These results are shown in FIG. 2
and a single component was detected in each case. These methods are
extremely sensitive in detecting microheterogeneity of
proteins.
The silver stain procedure involves silver-protein complex
formation. Immediately after electrophoresis, the gel was soaked in
400 ml of 40% (v/v) methanol, 10% (v/v) acetic acid for 60 min. and
then twice in 400 ml of 10% (v/v) ethanol, 5% (v/v) acetic acid,
each time 30 min. Subsequently, the gel was placed in 200 ml of an
oxidizer solution for 10 min., followed by a 30 min. wash with 400
ml deionized water. It was then treated with a silver reagent for
30 min., and two changes of a developer solution at 30 sec. and 5
min. The gel was finally developed in fresh developer for the
desired amount of time, and development was stopped by the addition
of 400 ml of 5% (v/v acetic acid. The oxidizer and developer
solutions are products of Bio-Rad Laboratories (Richmond,
Calif.).
Radioiodination was carried out using an iodination kit supplied by
New England Nuclear Corp. (Boston, Mass.). The kit contains
iodination beads coated with lactoperoxidase and glucose oxidase,
sodium phosphate buffer, 1% (w/v) .beta.-D-glucose for the
generation of hydrogen peroxide by the immobilized glucose oxidase,
and carrier free [.sup.125 I]-sodium iodide (1 to 2mCi). Labeling
was carried out at room temperature using Ep purified in Example 7.
The iodination mixture was subjected to Sephadex G100 gel
filtration to remove unincorporated .sup.125 I. A sample of 125I-Ep
with 10,000 cpm (1.5 ng) was analyzed by SDS-PAGE under the same
conditions as described in FIG. 1. Immediately after
electrophoresis, the gel was dried under vacuum and
autoradiographed on XAR-5 X-ray film.
FIG. 2A depicts a silver stained SDS-PAGE pattern: lane 1 shows Ep
from Example 7; lane 2 shows mol. weight standards. 2 .mu.g of
protein were loaded. FIG. 2B shows an autoradiograph of SDS-PAGE
patterns. Lane (1): molecular weight standards (4,000 cpm/band);
and lane (2): .sup.125 I-Ep from Example 7 (10,000 cpm).
The results of Ep purification of Examples 1, 6 and 7 are
summarized in following Table I:
TABLE 1
__________________________________________________________________________
Purification of Human Erythropoietin Units Sp. Activity Protein
Yield (%) Purification Factor Sample (u) (u/mg) (mg) Overall Step
Overall Step
__________________________________________________________________________
Urine con. 30,982 0.91 34,046 100 100 0 0 Phenyl- 25,378 124.4 204
82 82 137 137 Sepharose CL4B DIAC on 20,773 20,983 0.99 67 82
23,058 169 Sepharose- (Anti-Ep) RIAC on 18,309 32,122 0.57 59 88
35,299 1.53 Sepharose- (Anti-I)
__________________________________________________________________________
These values are based on the 2nd IRP standard (provided by WHO) as
mentioned above. It should be noted that at high levels of specific
activity, use of the IRP standard may result in an underestimate of
the actual specific activity, because the standard itself contains
many contaminating activities. For this reason, a purified Ep
standard which has been calibrated using the IRP standard at low
dose levels is often used. Because it is purer than the IRP, it
gives more accurate results than the IRP at high specific
activities.
When the activity of Ep prepared in Example 7 was reexamined using
purified sheep Ep as a standard, a specific activity of at least
66,000 .mu./mg was obtained.
In addition to the specific embodiments described above, numerous
other embodiments, variations, modifications and equivalents to the
present invention will be apparent to those of ordinary skill in
the art in light of the present specification, accompanying
drawings and appended claims.
* * * * *